Method for producing steviol and steviol glycoside using AOBGL3 homolog

Patent metadata

Field	Value
Publication number	US-10415071-B2
Application number	US-201615770896-A
Country	US
Kind code	B2
Filing date	Oct 28, 2016
Priority date	Oct 30, 2015
Publication date	Sep 17, 2019
Grant date	Sep 17, 2019

Abstract

The present invention provides a method for preparing a steviol glycoside and/or steviol comprising reacting glycosidase AOBGL1 and/or AOBGL3 or a variant thereof with a steviol glycoside having at least one monoglucoside bond and/or monoglucosyl ester bond, thereby cleaving said monoglucoside bond and/or monoglucosyl ester bond.

First claim

The invention claimed is:

1. A method for preparing a steviol glycoside and/or steviol comprising reacting a protein selected from the group consisting of proteins (a) to (c) shown below with a steviol glycoside having at least one monoglucoside bond and/or monoglucosyl ester bond, thereby cleaving said monoglucoside bond and/or monoglucosyl ester bond:
(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;
(b) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside; and
(c) a protein comprising the amino acid sequence of SEQ ID NO: 3 or 4.

2. The method according to claim 1 , wherein the steviol glycoside having at least one monoglucoside bond and/or monoglucosyl ester bond is selected from rebaudioside A, stevioside, rubusoside, steviolmonoside, and steviol monoglucosyl ester.

3. The method according to claim 2 , wherein the steviol glycoside to be prepared is selected from rebaudioside B and steviolbioside.

4. The method according to claim 1 , further comprising culturing a non-human transformant obtained by introducing a polynucleotide selected from the group consisting of polynucleotides (a) to (e) shown below into a host producing a steviol glycoside having at least one monoglucoside bond and/or monoglucosyl ester bond:
(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;
(c) a polynucleotide encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside;
(d) a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and which encodes a protein having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside, wherein highly stringent conditions include (i) 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, 50° C., (ii) 0.2×SSC, 0.1% SDS, 60° C.; 0.2×SSC, 0.1% SDS, 62° C., or (iii) 0.2×SSC, 0.1% SDS, 65° C.; and
(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 3 or 4.

5. The method according to claim 4 , wherein the polynucleotide is inserted into an expression vector.

6. The method according to claim 4 , wherein the transformant is transformed koji mold, transformed yeast, or a transformed plant.

7. The method according to claim 1 , wherein the protein is derived from a non-human transformed cell obtained by introducing, into a host cell, a polynucleotide selected from the group consisting of polynucleotides (a) to (e) shown below, with a steviol glycoside having at least one monoglucoside bond and/or monoglucosyl ester bond, thereby cleaving said monoglucoside bond and/or monoglucosyl ester bond:
(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;
(c) a polynucleotide encoding a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, and having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside;
(d) a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and which encodes a protein having an activity to cleave a monoglucoside bond and/or monoglucosyl ester bond of a steviol glycoside, wherein highly stringent conditions include (i) 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, 50° C., (ii) 0.2×SSC, 0.1% SDS, 60° C.; 0.2×SSC, 0.1% SDS, 62° C., or (iii) 0.2×SSC, 0.1% SDS, 65° C.; and
(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 3 or 4.

8. The method according to claim 7 , wherein the polynucleotide is inserted into an expression vector.

9. The method according to claim 7 , wherein the transformed cell is transformed koji mold, a transformed bacterium, or transformed yeast.

10. The method according to claim 7 , wherein the steviol glycoside having at least one monoglucoside bond and/or monoglucosyl ester bond is selected from rebaudioside A, stevioside, rubusoside, steviolmonoside, and steviol monoglucosyl ester.

11. The method according to claim 10 , wherein the steviol glycoside to be prepared is selected from rebaudioside B and steviolbioside.

12. The method according to claim 1 , further comprising reacting a protein selected from the group consisting of proteins (d) to (f) shown below, and a steviol glycoside having an unbranched β-1,2-glucoside bond:
(d) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 8;
(e) a protein having an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8, and having an activity to cleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and
(f) a protein comprising the amino acid sequence of SEQ ID NO: 7 or 8.

13. The method according to claim 12 , wherein the steviol glycoside having the unbranched β-1,2-glucoside bond comprises one or more steviol glycosides selected from rebaudioside D, rebaudioside E, stevioside, and steviolbioside.

Assignees

• Suntory Holdings Ltd

Inventors

• Ochiai Misa
• Ono Eiichiro

Classifications

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12N9/24

  acting on glycosyl compounds (3.2) · CPC title

• C12P15/00

  Preparation of compounds containing at least three condensed carbocyclic rings {(gibbanes C12P27/00; naphthacenes C12P29/00)} · CPC title

• C12P7/02

  containing a hydroxy group · CPC title

• C07H3/02

  Monosaccharides · CPC title